Rabbit anti hur antibody

BMSCs or primary osteoblasts were lysed in buffer (150 mmol·L⁻¹ NaCl, 50 mmol·L⁻¹ Tris, pH 7.8, 10% glycerol, 1 mmol·L⁻¹ EGTA, 0.5% NP-40, 1 mmol·L⁻¹ EDTA, 1 mmol·L⁻¹ PMSF, 1x cocktail) on ice with vigorous shaking for 30 min. The lysates were subjected to Western blotting with the indicated antibody. The following antibodies were used in the study: rabbit anti-HuR antibody (1:1 000, Cell Signaling Technology, Cat. No. 12582, monoclonal), rabbit anti-Atf4 antibody (1:1 000, Cell Signaling Technology, Cat. No. 11815, monoclonal), and rabbit anti-Gapdh antibody (1:5 000, Abways, Cat. No. AB0036, monoclonal).

BMSCs and osteoblasts were seeded in 15 cm culture plates, harvested after reaching over 90% confluency, washed twice with ice-cold PBS and suspended in 1 mL of RNA immunoprecipitation (RIP) buffer (50 mmol·L⁻¹ Tris pH 7.4, 150 mmol·L⁻¹ NaCl, 0.5% Igepal, 2 mmol·L⁻¹ VRC, 1 mmol·L⁻¹ PMSF and 1× protease inhibitor cocktail) with RNase inhibitor (TaKaRa, 2313 A). After 30 min of incubation at 4 °C on a rotating wheel, the lysates were centrifuged at 1 000 × g for 10 min at 4 °C, and the supernatants were precleared with 20 μL of Protein G PLUS-agarose beads (Santa Cruz, sc-2002). The precleared supernatants were then equally divided into two parts and incubated with 30 μL of Protein G PLUS-agarose beads with rabbit anti-HuR antibody (1:300, Cell Signaling Technology, Cat. No. 12582, monoclonal) or rabbit IgG (1:500, Cell Signaling Technology, Cat. No. 3900, monoclonal) for 3 h at 4 °C, followed by washing three times with RIP buffer. Finally, TRIzol reagent was added to the precipitate, and RNA was isolated for real-time RT‒PCR. Primers used for template amplification are shown in Table S2.